Microscope system

ABSTRACT

A microscope system is characterized in comprising a transmission illumination optical system having a light source ( 11 ) and a condenser lens ( 13 ); a first dry objective ( 15   a ) having a magnification of from 20 or higher to 40 or lower and capable of viewing by at least one of a differential interference viewing method and a modulation contrast viewing method; and a second dry objective ( 15   b ) having a magnification of from 60 or higher to 100 or lower and capable of viewing by a differential interference viewing method; the first objective ( 15   a ) and the second objective ( 15   b ) being exchangeable.

This is a continuation of PCT International Application No.PCT/JP2009/050555, filed on Jan. 16, 2009, which is hereby incorporatedby reference. This application also claims the benefit of JapanesePatent Application No. 2008-012823, filed in Japan on Jan. 23, 2008,which is hereby incorporated by reference.

TECHNICAL FIELD

The present invention relates to a microscope system for use withmicro-insemination, the system being capable of viewing using adifferential interference viewing method and a modulation contrastviewing method.

TECHNICAL BACKGROUND

At present, ICSI (intra-cytoplasmic sperm injection) is widely used as amicro-insemination method. In micro-insemination, a sperm is selectedusing a modulation contrast viewing method (see Patent Document 1, forexample), and a sperm having satisfactory motility and morphology isinjected into an ovum. However, recent advances in IVF (in-vitrofertilization) research have shown statistically that such factors asthe presence, size, and number of vacuoles in the sperm head aresignificantly related to the IVF success rate, but vacuoles in the spermhead are difficult to view by the modulation contrast viewing methodused for conventional ICSI. Therefore, a microscope system has beenproposed for enabling IMSI (intra-cytoplasmic morphologically selectedsperm injection, which is a micro-insemination method in which a spermis selected under high magnification), in which micro-insemination isperformed after selection by detailed viewing of the inside of the spermhead, to be performed in addition to the conventional ICSI. For example,a configuration is adopted in which the modulation contrast viewingmethod used in ICSI is employed jointly with a differential interferenceviewing method (see Patent Documents 2 and 3, for example) through ahigh-magnification objective that is used in IMSI.

Patent Document 1: Japanese Laid-open Patent Publication No. S51-128548

Patent Document 2: Japanese Patent No. 3456252

Patent Document 3: Japanese Patent No. 3415294

DISCLOSURE OF THE INVENTION Problems to be Solved by the Invention

In the differential interference viewing method used in themicro-insemination described above and well as in other fields ofbiological microscopy, structures must be observable in as much detailas possible, and an immersion objective having a high numerical aperture(NA) has generally been used as a high-magnification lens. As a result,in the conventional microscope system, an immersion-type lens is used asa high-magnification objective, and a dry-type lens is used as amedium-low-magnification objective, and during the switch to thedry-type medium-low-magnification objective for ICSI viewing after IMSIviewing through the immersion-type high-magnification objective, theimmersion liquid significantly interferes with the workability of IVF.In order to overcome this problem, a system has been proposed in whichan immersion objective is used as the medium-low-magnification objectivefor ICSI viewing, as with the high-magnification objective. In thissystem, however, the viscosity of the immersion liquid causes the sample(usually a dish) to move when immersion objectives are usedexchangeably, the sperm selected using the high-magnification objectivemay move out of view, and bubbles and the like are prone to beintroduced into the immersion liquid. These problems can make ICSIviewing extremely difficult after the objectives are exchanged.

The present invention was developed in view of such problems, and anobject of the present invention is to provide a microscope systemsuitable for IMSI/ICSI, whereby the sequence of operations formicro-insemination can be accurately and rapidly performed whilemaintaining resolving power, by viewing and selecting a sperm by adifferential interference viewing method using a dry-typehigh-magnification objective, then injecting the selected sperm into anovum by a differential interference viewing method or a modulationcontrast viewing method using an exchanged low-magnification objective,which is also a dry-type objective.

Means to Solve the Problems

In order to achieve such objects as those described above, the presentinvention is a microscope system suitable for micro-insemination, and ischaracterized in comprising a transmission illumination optical systemhaving a light source and a condenser lens; a first dry objective havinga magnification of from 20 or higher to 40 or lower and capable ofviewing by at least one of a differential interference viewing methodand a modulation contrast viewing method; and a second dry objectivehaving a magnification of from 60 or higher to 100 or lower and capableof viewing by a differential interference viewing method; the firstobjective and the second objective being exchangeable.

Advantageous Effects of the Invention

As described above, according to the present invention, a microscopesystem suitable for micro-insemination can be provided whereby thesequence of operations in micro-insemination can be rapidly andaccurately performed with satisfactory workability while the appropriateresolving power is maintained.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic sectional view showing the microscope systemutilizing a differential interference viewing method according to thepresent embodiment;

FIG. 2 is a view showing the underlying principle of the modulationcontrast viewing method, which is another viewing method used in themicroscope system of the present embodiment;

FIG. 3 is a view showing the positional relationship between theaperture image and the modulator in the modulation contrast viewingmethod according to the present embodiment, wherein FIG. 3A, FIG. 3B,and FIG. 3C correspond to FIG. 2A, FIG. 2B, and FIG. 2C;

FIG. 4A is a view showing an example of the shape of the sample, andFIG. 43 is a view showing the shading that appears corresponding to thesample in the modulation contrast viewing method according to thepresent embodiment;

FIG. 5 is a view showing the MTF curve of the incoherent optical systemaccording to the present embodiment;

FIG. 6 is a view showing the contrast MTF curves of phase samples in thedifferential interference viewing method according to the presentembodiment;

FIG. 7 is a sectional view showing the structure of the second objective(dry-type high-magnification objective) according to a first example;and

FIG. 8 shows several aberration diagrams for the second objectiveaccording to the first example, wherein FIG. 8A is a sphericalaberration diagram, FIG. 8B is an astigmatism diagram, and

FIG. 8C is a distortion diagram.

EXPLANATION OF NUMERALS AND CHARACTERS

-   -   11 light source (transmission illumination optical system)    -   12 collector lens    -   13 condenser lens (transmission illumination optical system)    -   14 sample    -   15 objective    -   15 a medium-magnification objective (first objective) (capable        of viewing by a differential interference viewing method)    -   15 b high-magnification objective (second objective)    -   16 turret    -   BP1 illumination-side birefringent optical member    -   BP2 imaging-side birefringent optical member    -   P polarizer    -   A analyzer    -   22 medium-magnification objective (first objective) (capable of        viewing by a modulation contrast viewing method)    -   23 aperture plate    -   23 a rectangular aperture    -   24 modulator

DESCRIPTION OF THE PREFERRED EMBODIMENTS

Preferred embodiments will be described with reference to the drawings.FIG. 1 is a schematic sectional view showing a microscope systemsuitable primarily for micro-insemination, according to the presentembodiment. The microscope system according to the present embodiment issuitable for IMSI (intra-cytoplasmic morphologically selected sperminjection, which is a micro-insemination method in which a sperm isselected under high magnification) and ICSI (intra-cytoplasmic sperminjection), and as shown in FIG. 1, the microscope system has atransmission illumination optical system composed of a light source 11and a condenser lens 13; a collector lens 12; a sample 14; an objective15; a turret 16; an illumination-side birefringent optical member BP1;an imaging-side birefringent optical member BP2; a polarizer P; and ananalyzer A.

In FIG. 1, the illuminating light from the light source 11 is incidenton the polarizer P after being collected by the collector lens 12, andis converted to linearly polarized light. The illumination-sidebirefringent optical member BP1 and the condenser lens 13 forirradiating the illuminating light on the sample 14 are provided inorder from the light source 11 side in the optical path between thepolarizer P and the sample 14. The linearly polarized light emitted fromthe polarizer P is incident on the illumination-side birefringentoptical member BP1 and split by birefringence into two linearlypolarized light components having mutually orthogonal directions ofvibration, and the linearly polarized light components are incident onthe condenser lens 13. The two rays split by the illumination-sidebirefringent optical member BP1 travel at a small separation angle α,are converted to parallel rays separated from each other at a smallshear distance S by the collecting effect of the condenser lens 13, andilluminate the sample 14. The two rays transmitted at slightly separatedpositions on the sample 14 are incident on the imaging-side birefringentoptical member BP2 via the objective 15 and combined by the birefringenteffect of the imaging-side birefringent optical member BP2 so as totravel on the same optical path. The combined rays are incident on theanalyzer A, the analyzer A extracts only the components of the mutuallyorthogonal linearly polarized light that are vibrating in the samedirection, and these components interfere. As a result, a magnifiedimage (interference image 17) on the image plane is formed by aninterference fringe that forms according to the phase differenceimparted between the two light rays as the rays pass through the sample14 in slightly different positions. An observer can view the magnifiedimage 17 through an eyepiece optical system not shown in the drawing.

When the sample 14 is planar and homogeneous, (since there is no phasedifference between the two split rays of light,) the magnified image 17is an image that has a uniform intensity distribution and is devoid ofcontrast. On the other hand, when the sample 14 is heterogeneous and hasgradients and level differences, (since there is a phase differencebetween the two split rays of light,) contrasts occur in the magnifiedimage 17 in portions where the refractive index varies, or in portionsthat correspond to gradients and level differences. The refractive indexvariations or the gradients and level differences are thereby madevisible, and the sample 14 can be viewed at magnification.

The objective 15 is composed of a first objective 15 a having amagnification of from 20 or higher to 40 or lower (hereinafter referredto as a dry-type medium-magnification objective or amedium-magnification objective), and a second objective 15 b having amagnification of from 60 or higher to 100 or lower that is capable ofcontrast viewing by a differential interference viewing method(hereinafter referred to as a dry-type high-magnification objective or ahigh-magnification objective), and the medium-magnification objective 15a and the high-magnification objective 15 b are configured so as to beexchangeable with the aid of a turret 16 or the like.

The first objective 15 a is preferably capable of viewing by at leastone of the abovementioned differential interference viewing method ormodulation contrast viewing method. The underlying principles of themodulation contrast viewing method will be briefly described using FIGS.2 through 4. In the drawings, the reference numeral 21 refers to acondenser lens, S refers to a sample, 22 refers to an objective, 23refers to an aperture plate, and 24 refers to a disk-shaped modulator.The aperture plate 23 has a rectangular aperture 23 a positioned at adistance from the center, in the focal position on the light source sideof the condenser lens 21. The modulator 24 is provided in a positionsubstantially conjugate to the aperture plate 23, and a 100%transmittance region 24 a that may include the image of the aperture 23a, a region 24 b of 15% transmittance, for example, and a 0%transmittance region 24 c are formed in the stated order adjacent toeach other in the modulator 24.

In this optical system, since the rectangular aperture 23 a is disposedin an eccentric position with respect to the optical axis, light that isincident on the condenser lens 21 is emitted so as to illuminate thesample S at an oblique angle. When the transparent sample S is planar asshown in FIG. 2A, the flux of light passing through the sample S isfocused in the region 24 b of the modulator 24 by the objective 22, andan aperture image 23 a′ is formed in the region 24 b, as shown in FIG.3A. When the surface of the sample S is inclined so as to rise to theright, as shown in FIG. 2B, the flux of light that passes through thesample S is refracted to the right and focused in the region 24 c of themodulator 24, and the aperture image 23 a′ is formed in the region 24 c,as shown in FIG. 3B. When the surface of the sample S is inclined so asto rise to the left, as shown in FIG. 2C, the flux of light that passesthrough the sample S is refracted to the left and focused in the region24 a of the modulator 24, and the aperture image 23 a′ is formed in theregion 24 a, as shown in FIG. 3C.

As is apparent from this description, when the sample S is a colorlesstransparent body having flat surfaces and inclined surfaces such asshown in FIG. 4A, the viewed image is such that the flat portions appeargray and the inclined portions appear black or white such as shown inFIG. 4B. The modulation contrast viewing method thus enables even acolorless transparent sample to be viewed as a three-dimensional imagewith shading, through the effects of focal illumination and regions ofthe modulator 24 having different transmittances.

In this microscope system, when the medium-magnification objective 15 athat is capable of contrast viewing by a modulation contrast viewingmethod is used, a change is made to the illumination-side birefringentoptical member BP1, imaging-side birefringent optical member BP2,polarizer P, and analyzer A, which are members used for viewing by thedifferential interference viewing method described above; and theaperture plate 23 and the modulator 24 are placed between the lightsource 11 and the condenser lens 13 (in the stated order from the lightsource 11).

In the microscope system according to the present embodiment, thefollowing Conditional Expressions (1) and (2) are preferably satisfied,where NA is the numerical aperture of the dry-type second objective(high-magnification objective), f is the focal distance, and MD is theworking distance.

0.78≦NA<1.0  (1)

f/3≦WD<2f  (2)

In viewing by the differential interference viewing method using thesecond objective, the following Conditional Expression (3) is preferablysatisfied, where S is the shear distance in the object plane, NA is thenumerical aperture of the second objective, and λ is the wavelength ofthe viewed light.

0.3≦S≦0.61λ/NA  (3)

In the past, resolving ability was considered critical in viewing by thedifferential interference viewing method using a high-magnification (60or higher and 100 or lower), high numerical aperture immersion objectivein the field of biological microscopy, and intervention using videoenhancement or other image processing was assumed. However, during IMSIor other micro-insemination, visual observation is consistently used inorder to rapidly and accurately perform the sequence of operationswhereby a more satisfactory sperm is selected from a wide range of(numerous) sperm under high magnification, and the selected sperm isthen injected into an ovum under medium magnification. There isaccordingly a need for visually adequate contrast, but optimumconditions have not yet been presented for an objective capable ofviewing by a differential interference viewing method that would satisfythe need for adequate contrast. The optimum Conditional Expressions (1)through (3) have therefore been derived for the second objective(high-magnification objective) for enabling viewing by the differentialinterference viewing method in the present microscope system. Therefollows a description of Conditional Expressions (1) through (3) in thestated order.

Conditional Expression (1) for specifying the range of appropriatenumerical apertures NA of the high-magnification objective will first bedescribed. In the present microscope system, the head portion of asperm, an image of which is viewed in the present microscope system, isabout 4 to 5 μm in size, and it has been confirmed that one to tenvacuoles of various sizes are scattered through the head portion in asingle focal plane. Consequently, the ability to see and identifydetails about 0.4 to 0.5 μm in size with good contrast is understood tobe adequate for IMSI applications.

FIG. 5 shows the MTF curve (i.e., the relationship between contrast onthe vertical axis and the resolving power of the optical system on thehorizontal axis) of the incoherent optical system commonly used in amicroscope optical system. On the horizontal axis f/f0 of FIG. 5, thespatial frequency f is normalized so that f0=NA/λ, where f is thespatial frequency, NA is the numerical aperture of the objective, and λis the wavelength of the viewed light. The relationship to thehorizontal resolving power RES corresponding to the spatial frequency fis indicated by Equation (4), where RES is the horizontal resolvingpower of the microscope optical system.

RES=1/f={1/(f/f0)}×{λ/NA}  (4)

The maximum value of 2.0 for the horizontal axis f/f0 shown in FIG. 5corresponds to the maximum resolving power RES(max) of the microscopeoptical system. Therefore, by substituting f/f0=2.0 in Equation (4),Equation (5) is obtained, in which the maximum resolving power RES(max)of the microscope optical system is indicated. An image is difficult tosee when there is no additional margin beyond the numerical aperture NAthat corresponds to the maximum resolving power RES(max) specified byConditional Expression (5).

RES(max)={1/2.0}×{λ/NA}=0.5λ/NA  (5)

It is known that the human eye generally has difficulty distinguishingcontrast values of 0.1 or lower, and that visibility is satisfactorywhen the contrast value is 0.2 or higher. In FIG. 5, since the spatialfrequency f/f0 at which the contrast value is 0.1 is near 1.6, Equation(6) must be satisfied by substituting these values into Equation (4) inorder for the horizontal resolving power RES to be 0.4 μm (the size of avacuole in the sperm head as the viewed image).

RES={1/1.6}×{λ/NA}=0.4  (6)

Since visual observation is assumed in IMSI and ICSI, which are theapplications for which the present microscope system is used, thecentral wavelength λ during viewing is preferably in the vicinity of 500nm to 550 nm when visibility to the eye is considered. The reason forthis is that 500 nm and 550 nm are visibility peaks for dark locationsand bright locations, respectively. When the central wavelength λ of 500nm=0.5 μm for viewing is substituted into Equation (6), Equation (7)below is obtained as the lower limit of the numerical aperture NArequired for the high-magnification objective.

NA={1/1.6}×0.5 [μm]/0.4 [μm]=0.78  (7)

Since the spatial frequency f/f0 at which the contrast value is 0.2,which is considered to produce good visibility, is near 1.4 according toFIG. 5, a more preferred lower limit for the numerical aperture NA isobtained from Equation (8) by substituting into Equation (6) in themanner described above.

NA={1/1.4}×0.5 [μm]/0.4 [μm]=0.89  (8)

The numerical aperture NA is indicated by NA=n·sin(φ/2), where n is therefractive index of the medium between the objective and the sample, andφ is the aperture angle, and the medium in the present embodiment is air(refractive index n=1). Therefore, the maximum numerical aperture NA is1.

In summary, in the microscope system of the present embodiment, therange of the numerical aperture NA of the high-magnification objectivepreferred for enabling visual observation with good contrast isexpressed by Conditional Expression (1), i.e., 0.78≦NA<1.0. The range ofthe numerical aperture NA of the high-magnification objective is morepreferably 0.89≦NA<1.0, according to Conditional Expression (8).

Conditional Expression (2) will next be described, which specifies theappropriate range of the working distance WD in the high-magnificationobjective. In the conventional microscope system, a large workingdistance is generally difficult to obtain with thehigh-numerical-aperture immersion objective used as thehigh-magnification objective. Therefore, when such an objective is usedin ICSI viewing or IMSI viewing, which require that the sample be keptat 37° C. by an insulating device or the like during working, theinsulation device and the distal end of the objective are prone tointerfere during the switch from high magnification to themedium-magnification objective. Since a large working distance cannot beobtained, only the area immediately below the cover glass can be viewedby the high-numerical-aperture immersion objective, and sperm that arepositioned at a distance from the cover glass cannot be targeted forselection. Therefore, a dry-type lens is used as the high-magnificationobjective in the present microscope system, and the condition of havingthe large working distance WD indicated by Conditional Expression (2) isthereby provided in addition to the condition of the numerical apertureNA indicated by Conditional Expression (1).

One factor that determines the working distance is the magnification ofthe objective. In an infinity-corrected optical system, themagnification of the objective is determined by the ratio of the focaldistances of the imaging lens and the objective (in a high-magnificationobjective having a magnification of from 60 or higher to 100 or lowersuch as in the present embodiment, when the focal distance of theimaging lens is 200 μm, for example, the focal distance of the objectiveis 2.00 to 3.33 m), and the focal distance shortens as the magnificationincreases. In general, since the working distance is proportional to thefocal distance of the objective, the working distance decreases as themagnification of the objective increases. Another factor that determinesthe working distance is the numerical aperture. When the size of thenumerical aperture is the same, a longer working distance corresponds toa greater height of the light rays at the first lens surface (surface ofthe lens on the object side) of the objective, and aberration becomesmore difficult to correct.

In other words, since increasing the working distance of the objectiveis incompatible with increasing the magnification and numerical aperturethereof, high-magnification objectives used in conventional microscopesystems are polarized between those with an emphasis on working distanceand those with an emphasis on magnification and numerical aperture.Specifically, magnification 60/numerical aperture 0.7/working distance 2mm are typical specifications for working-distance-oriented objectives;and magnification 100/numerical aperture 1.4/working distance 0.1 mm aretypical specifications for magnification/numerical-aperture-orientedobjectives. However, when viewing IMSI is the intended application, thenumerical aperture NA does not necessarily exceed 1, as indicated byConditional Expression (1), and increasing the working distance by acorresponding amount leads to enhanced working efficiency.

Therefore, Conditional Expression (2), i.e., f/3≦WD<2f, is preferablysatisfied in the present embodiment, where NA is the numerical apertureof the high-magnification objective (second objective), f is the focaldistance, and WD is the working distance. When Conditional Expression(2) is below the lower limit value, there is increased risk of suchproblems as interference between the distal end of the objective and theinsulation device during exchanging of the objectives. When ConditionalExpression (2) exceeds the upper limit value, the light rays at thefirst lens surface of the objective are too high, and it is difficult toensure the numerical aperture NA specified by Conditional Expression(1).

Conditional Expression (3) will next be described. ConditionalExpression (3) specifies the optimum range of the shear distance S inthe differential interference viewing method. FIG. 6 shows the phasecontrast MTF curves as the shear distance S is varied from 1.5λ/NA to0.15λ/NA (Patent Document 3 gives a detailed description of the methodfor computing the phase contrast MTF in the differential interferenceviewing method). In FIG. 6, on the horizontal axis f/f0, the spatialfrequency f is normalized by the reference frequency f0=NA/λ specifiedby the numerical aperture NA of the objective, and the vertical axisindicates the contrast MTF for the phase object at each frequency.

It is apparent from FIG. 6 that the phase contrast MTF curves in thedifferential interference viewing method show larger contrast valuesthan the incoherent MTF curve. It is also apparent from FIG. 6 that thephase contrast MTF has a negative value when the shear distance S islarge (e.g., 1.5λ/NA), and this negative value indicates a statereferred to as spurious resolution, in which black and white areinverted. A state in which spurious resolution does not occur, i.e., astate in which the phase contrast MTF is not negative, is generallyconsidered to be preferable in viewing by the differential interferenceviewing method.

Therefore, the limits of a low spatial frequency band (region in whichthe value of the horizontal axis f/f0 is small) that satisfies acondition whereby the phase contrast MTF value is not negative willfirst be described. It is apparent from FIG. 6 that the maximum sheardistance S of the low spatial frequency band is approximately 0.61λ/NAto 0.5λ/NA, and these values correspond precisely with the pointresolving power or line resolving power of the microscope opticalsystem. The curve for a shear distance S of 0.61λ/NA in FIG. 6 showsthat the contrast values are high in the low spatial frequency band, butthat the contrast value is about 0.1 in the vicinity of horizontal axisf/f0=1.4 in the high spatial frequency band, corresponding preciselywith the limit of visibility. The maximum value of the shear distance Sof the objective in the present embodiment is thus 0.61λ/NA, at whichthe point resolving power can be maintained. A more preferred maximumvalue for the shear distance S is 0.5λ/NA, (which is closer to theincoherent MTF curve than the curve for 0.61λ/NA,) at which the lineresolving power can be maintained.

The limits of the high spatial frequency band (region in which the valueof the horizontal axis f/f0 is large) that satisfies the conditionwhereby the phase contrast MTF is not negative will next be described.It is apparent from FIG. 6 that the maximum shear distance S of the highspatial frequency band corresponds to the curve having the highestcontrast in the vicinity of horizontal axis f/f0=1.6, i.e., the curvefor 0.3λ/NA. In this instance, instead of sacrificing contrast in thelow spatial frequency band to a certain degree, the visibility in thehigh spatial frequency band can be kept at 0.1, which is substantiallyequal to the contrast value of the incoherent MTF. When the sheardistance S is further reduced, contrast decreases in the low spatialfrequency band as well as in the high spatial frequency band, and isunsuitable for the purposes of the present embodiment. The minimum valueof the shear distance S for the objective in the present embodiment isthus 0.32λ/NA.

In summary, by setting a shear distance S that satisfies ConditionalExpression (3), i.e., 0.3λ/NA≦S≦0.61λ/NA, and more preferably0.32λ/NA≦S≦0.5λ/NA, vacuoles in the sperm head can be visualized withhigh contrast and with almost no compromise to the resolving power ofthe objective in a differential interference viewing method.

The second objective (high-magnification objective) according to thepresent embodiment preferably has a correction ring for correctingaberration fluctuation due to changes in temperature, cover glassthickness, and other factors. This is because the use of a correctionring makes it possible to eliminate aberration caused by temperature,error in the cover glass thickness, and other factors; and to makeadjustments so that the resolution and contrast of the objective areboth maximized.

EXAMPLES

Examples of the second objective (dry-type high-magnification objective)according to the present embodiment will be described.

First Example

A first example will be described using FIG. 7, FIG. 8, and Table 1.FIG. 7 is a sectional view showing the lens structure of the secondobjective (dry-type high-magnification lens) according to the presentexample. As shown in FIG. 7, the microscope objective in the presentexample comprises, in order from the object, a positive meniscus lens L1having a concave surface facing the object; a positive meniscus lens L2having a concave surface facing the object; a cemented lens composed ofa double-concave lens L3 and a double-convex lens L4; a cemented lenscomposed of a double-concave lens L5 and a double-convex lens L6; acemented lens composed of a planoconcave lens L7 and a double-convexlens L8; a double-convex lens L9; a cemented lens composed of a negativemeniscus lens L10 having a concave surface facing the object, adouble-convex lens L11, and a negative meniscus lens L12 having aconcave surface facing the object; a cemented lens composed of adouble-convex lens L13 and a double-concave lens L14; and a cementedlens composed of a double-concave lens L15 and a double-convex lens L16.A cover glass C is provided on the object side of the positive meniscuslens L1.

Table 1 shows the various values of the lenses that constitute thesecond objective of the present example. In the various entries shown inTable l, m represents the order of lens surfaces (hereinafter referredto as surface numbers) from the object along the direction of travel ofa ray of light, r represents the radius of curvature of each lens, drepresents the distance on the optical axis from each optical surface tothe next optical surface (or image surface), nd represents therefractive index with respect to the d-line (wavelength: 587.6 nm), andνd represents the Abbe number based on the d-line. Surface numbers 1through 25 in Table 1 correspond to surfaces 1 through 25 shown in FIG.7. In the table, β represents the magnification, WD represents theworking distance, and NA represents the numerical aperture.

In the table, the radius of curvature r, the distance d to the next lenssurface, and other lengths are generally represented in millimeterunits. However, since equivalent optical performance is obtained whetherin proportional magnification or proportional reduction in the opticalsystem, the units are not limited to millimeters; other appropriateunits may be used. The value “∞” for the radius of curvature in thetable indicates a plane, and the refractive index of “1.00000” for airis not noted.

TABLE 1 [Lens data] β = 100, WD = 1.4, NA = 0.85 m r d nd νd ∞ 0.170001.52216 58.80 (cover glass C) ∞ 2.50462 1 −6.47161 2.37000 1.8160046.621 2 −4.72849 0.10000 3 −83.0402 2.83000 1.49782 82.557 4 −10.66070.15000 5 −46.5266 1.00000 1.61340 44.266 6 24.27074 4.95000 1.4338595.247 7 −14.7782 0.20000 8 −174.834 1.00000 1.61340 44.266 9 24.114954.95000 1.43385 95.247 10 −14.5394 0.20000 11 ∞ 1.00000 1.61340 44.26612 28.67355 4.20000 1.43385 95.247 13 −23.0153 0.20000 14 48.935483.00000 1.49782 82.557 15 −65.8669 1.52002 16 21.78198 1.00000 1.7291654.660 17 11.99437 6.30000 1.49782 82.557 18 −12.5334 1.20000 1.7550052.318 19 −59.9845 7.75003 20 27.89895 3.35000 1.59240 68.328 21−7.03528 8.40000 1.65412 39.682 22 5.87805 1.40000 23 −4.44814 1.000001.80440 39.567 24 11.0118 1.90000 1.92286 18.896 25 −11.4804

FIG. 8 shows several aberration diagrams for the microscope objectiveaccording to the present example, wherein FIG. 8A is a sphericalaberration diagram, FIG. 8B is an astigmatism diagram, and

FIG. 8C is a distortion diagram. In FIG. 8, NA is the numericalaperture, y is the image height (mm), the solid line is the d-line(wavelength: 587.6 nm), the dashed line is the C-line (wavelength: 656.3nm), the single-dashed line is the F-line (wavelength: 486.1 nm), andthe double-dashed line is the g-line (wavelength 435.8 nm). In theastigmatism diagram, the solid line represents the sagittal imagesurface, and the dashed line represents the meridional image surface.

As is apparent from the aberration diagrams shown in FIG. 8, aberrationsare satisfactory corrected, and excellent imaging performance ismaintained in the second objective (dry-type high-magnificationobjective) according to the present example.

As described above, according to the present invention, there isprovided a microscope system suitable for IMSI/ICSI, whereby it ispossible to accurately and rapidly perform the sequence of operations inwhich the presence of vacuoles in a sperm head and other characteristicsare viewed by a differential interference viewing method using adry-type high-magnification (60 or higher and 100 or lower) objective toselect a sperm, whereupon the objectives are exchanged through the useof the turret 16 or the like, and the selected sperm is injected into anovum while viewed by a differential interference viewing method ormodulation contrast viewing method using a medium-magnification (20 orhigher and 40 or lower) objective, which is also a dry-type objective.

The essential characteristics of embodiments were described above to aidin understanding the present invention, but the present invention shallnot be construed as being limited to the embodiments described above.

1-8. (canceled)
 9. A microscope system for micro-insemination,comprising: a transmission illumination optical system having a lightsource and a condenser lens; a first dry objective having amagnification of from 20 or higher to 40 or lower and capable of viewingby at least one of a differential interference viewing method and amodulation contrast viewing method; and a second dry objective having amagnification of from 60 or higher to 100 or lower and capable ofviewing by a differential interference viewing method; the firstobjective and the second objective being exchangeable.
 10. Themicroscope system for micro-insemination according to claim 9,characterized in that the following conditional expressions aresatisfied:0.78≦NA<1.0f/3≦WD<2f, where NA is the numerical aperture of the second objective, fis focal length thereof, and WD is working distance thereof.
 11. Themicroscope system for micro-insemination according to claim 9,characterized in that the following conditional expression is satisfiedwhen viewing by the differential interference viewing method using thesecond objective:0.3λ/NA≦S≦0.61λ/NA, where S is the shear distance in the object plane,NA is the numerical aperture of the second objective, and λ is thewavelength of the viewed light.
 12. The microscope system formicro-insemination according to claim 9, characterized in that thesecond objective has a correction ring for correcting aberrationfluctuation due to changes in factors including temperature and coverglass thickness.